We propose to continue our studies of the replication of bacteriophage lambda DNA in an in vitro system that is reconstituted with 20 highly purified lambda and E. coli proteins. Our long range goal is to achieve a detailed mechanistic understanding of the biochemical events that occur during the initiation, propagation, termination, and regulation of lambda DNA replication. The viral O and P replication proteins promote the assembly of an ordered series of nucleoprotein structures at the lambda replication origin (ori- lambda) prior to priming and DNA chain elongation. Recent findings indicate that the first two of these structures, the O-some and the ori- lambda.O.P.DnaB complex, have the capacity to capture and hold alternate DNA conformations induced by a combination of negative DNA supercoiling and O protein binding to ori lambda. Moreover, the step leading to formation of these "pre-open" complexes appears to be a key point of regulation of lambda DNA replication. We will characterize this step further (a) by defining its dependence on superhelical tension; (b) by defining the amino acid residues of the lambda O and P proteins and E. coli DnaB helicase that interact with single stranded DNA and/or with the A/T-rich region of ori lambda; and (c) by analyzing how transcriptional events distant from orilambda facilitate the DNA melting step. Using radiolabeled proteins, we will define the stoichiometries of the O, P, and DnaB proteins in pre-initiation structures assemble at orilambda. The fate of radiolabeled O, P and DnaB following initiation will be monitored. We will continue our efforts to use x-ray crystallography to define the tertiary structure of the DNA-binding domain of the lambda O initiator. In related experiments, we will use a genetic approach to define the amino acid residues of the N-terminal domain of O involved in specific recognition of origin DNA sequences. We will also select mutant orilambda DNA replication and identify the molecular step or steps in the initiation pathway affected by individual mutations. Biochemical studies of lambda DNA replication will continue to provide important insights into the biological mechanisms used in the initiation and regulation of chromosomal DNA replication. This knowledge will help guide studies of these vital processes in more complex organisms.